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Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M 3 muscarinic receptors
Author(s) -
Yang Chuen Mao,
Yo YingLing,
Wang YenYi
Publication year - 1993
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1993.tb13910.x
Subject(s) - carbachol , muscarinic acetylcholine receptor , medicine , endocrinology , muscarinic antagonist , atropine , calcium , chemistry , antagonist , stimulation , muscarinic acetylcholine receptor m3 , egta , fura 2 , biology , receptor , cytosol , biochemistry , enzyme
1 The regulation of cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura‐2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose‐dependent rise in [Ca 2+ ] i which was followed by a stable plateau phase. The EC 50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 μ m , respectively. 2 Atropine (10 μ m ) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca 2+ ] i within 5 s. Therefore, the changes in [Ca 2+ ] i by carbachol were mediated through the muscarinic AChRs. 3 AF‐DX 116 (a selective M 2 antagonist) and 4‐diphenylacetoxy‐ N ‐methylpiperidine (4‐DAMP, a selective M 3 antagonist) inhibited the carbachol‐stimulated increase in [Ca 2+ ] i with p K B values of 6.4 and 9.4, respectively, corresponding to low affinity for AF‐DX 116 and high affinity for 4‐DAMP in antagonizing this response. 4 The plateau elevation of [Ca 2+ ] i was dependent on the presence of external Ca 2+ . Removal of Ca 2+ by the addition of 2 m m EGTA caused the [Ca 2+ ] i to decline rapidly to the resting level. In the absence of external Ca 2+ , only an initial transient peak of [Ca 2+ ] i was seen which then declined to the resting level; the sustained elevation of [Ca 2+ ] i could then be evoked by the addition of Ca 2+ (1.8 m m ) in the continued presence of carbachol. 5 Ca 2+ influx was required for the changes of [Ca 2+ ] i , since the Ca 2+ ‐channel blockers, diltiazem (10 μ m ), nifedipine (10 μ m ), verapamil (10 μ m ) and Ni 2+ (5 m m ), decreased both the initial and sustained elevation of [Ca 2+ ] i in response to carbachol. These Ca 2+ ‐channel blockers also decreased the sustained elevation of [Ca 2+ ] i when applied during the plateau phase. 6 In conclusion, we have demonstrated that the initial detectable increase in carbachol‐stimulated [Ca 2+ ] i is due to the release of Ca 2+ from internal stores, followed by the flux of external Ca 2+ into the cells. This influx of extracellular Ca 2+ partially involves an L‐type Ca 2+ ‐channel. M 3 muscarinic receptors appear to mediate the Ca 2+ mobilization in canine TSMCs.